Abstract:

BACKGROUND: Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and
animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development
and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in
structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that
controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this
study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to
determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin
expression was characterized in ticks experimentally infected with the cattle pathogen, Anaplasma marginale.
Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with
Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA
interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by
capillary feeding (CF).
RESULTS: Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick
vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick
species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis
infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with
Francisella tularensis increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF,
subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was
upregulated in D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce
subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown
mechanism.
CONCLUSION: Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick
innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and
consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a
role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate
immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional
support for the role of subolesin in other molecular pathways including those required for tissue development and
function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that
subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that
interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin
knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular
interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control
of global gene expression in ticks.